Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Pathogen infection and infestation can lead to significant health issues in humans, animals and plants.
Crop losses due to plant pathogens such as fungal pathogens, for example, are a major problem in the agricultural industry and each year millions of dollars are spent on the application of fungicides to curb these loses (Oerke and Dehne (2004) Crop Protection 23:275-285).
Although chemical pathogenicides have been successful in human and veterinary medicine and in the agricultural sector, there is a range of environmental and regulatory concerns with the continued use of chemical agents to control pathogen infection and infestation. The increasing use of these agents is also providing selective pressure for emergence of resistance in pathogen species. This is of particular concern in relation to the widespread use of antibiotics to treat infection in humans and animals. There is clearly a need to develop alternative mechanisms of controlling infection and infestation in humans, animals and plants by pathogens. This need extends to controlling pathogen contamination in soil and other environmental sites to which humans, animals and plants are exposed.
Humans, animals and plants have evolved various systems to provide some natural protection against pathogen infection and infestation. Whilst innate immune mechanisms have been studied in relation to the species investigated, little is known about the use of components of these systems across different species. In plants, these components include small, disulfide-rich proteins which play a large role in both the constitutive and inducible aspects of plant immunity. They can be categorized into families based on their cysteine arrangements and include the thionins, snakins, thaumatin-like proteins, hevein- and knottin-type proteins, lipid transfer proteins, α-hairpinins and cyclotides as well as defensins.
Plant defensins are small (45-54 amino acids), basic proteins with four to five disulfide bonds (Janssen et al. (2003) Biochemistry 42(27):8214-8222). They share a common disulfide bonding pattern and a common structural fold, in which a triple-stranded, antiparallel β-sheet is tethered to an α-helix by three disulfide bonds, forming a cysteine-stabilized αβ motif A fourth disulfide bond also joins the N- and C-termini leading to an extremely stable structure. A variety of functions has been attributed to defensins, including anti-bacterial activity, protein synthesis inhibition and α-amylase and protease inhibition (Colilla et al. (1990) FEBS Lett 270(1-2):191-194; Bloch and Richardson (1991) FEBS Lett 279(1):101-104). Plant defensins have been expressed in transgenic plants, resulting in increased resistance to target pathogens. For example, potatoes expressing the alfalfa defensin (MsDefl, previously known as alfAFP) showed significant resistance against the fungal pathogen Verticillium dahliae compared to non-transformed controls (Gao et al. (2000) Nat Biotechnol 18(12):1307-1310). Expression of a Dahlia defensin (DmAMP1) in rice was sufficient to provide protection against two major rice pathogens, Magnaporthe oryzae and Rhizoctonia solani (Jha et al. (2009) Transgenic Res 18(0:59-69).
The structure of defensins consists of seven ‘loops’, defined as the regions between cysteine residues. Loop 1 encompasses the first β-strand (1A) as well as most of the flexible region that connects this β-strand to the α-helix (1B) between the first two invariant cysteine residues. Loops 2, 3 and the beginning of 4 (4A) make up the α-helix, while the remaining loops (4B-7) make up β-strands 2 and 3 and the flexible region that connects them (van der Weerden et al. (2013) Cell Mol Life Sci 70(19): 3545-3570).
Despite their conserved structure, plant defensins share very little sequence identity, with only the eight cysteine residues completely conserved. The cysteine residues are commonly referred to as “invariant cysteine residues”, as their presence, location and connectivity are conserved amongst defensins. Based on sequence similarity, plant defensins can be categorized into different groups. Within each group, sequence homology is relatively high whereas inter-group amino acid similarity is low (van der Weerden et al. (2013) Cell Mol Life Sci 70(19): 3545-3570).
There are two major classes of plant defensins. Class I defensins consist of an endoplasmic reticulum (ER) signal sequence followed by a mature defensin domain. Class II defensins are produced as larger precursors with C-terminal pro-domains or pro-peptides (CTPPs) of about 33 amino acids. Most of the Class II defensins identified to date have been found in Solanaceous plant species.
Class II Solanaceous defensins are expressed in floral tissues. They include NaD1, which is expressed in high concentrations in the flowers of ornamental tobacco Nicotiana alata (Lay et al. (2003) Plant Physiol 131(3):1283-1293). The anti-fungal activity of this peptide involves binding to the cell wall, permeabilization of the plasma membrane and entry of the peptide into the cytoplasm of the hyphae (van der Weerden et al. (2008) J Biol Chem 283(21):14445-14452) and induction of reactive oxygen species (Hayes et al. (2014) Cell Mol Life Sci. February 2014, on line ISSN 1420-682X).
Expression of NaD1 in cotton enhances the resistance to the fungal pathogens Fusarium oxysporum fsp. vasinfectum and Verticillium dahliae. Under field conditions, plants expressing NaD1 are twice as likely to survive compared to untransformed control plants and the lint yield per hectare is doubled. Despite this, there was still a significant level of disease in the NaD1-expressing plants (Gaspar et al. (2014) J Exp Bot February 6 epub).
Class II Solanaceous defensins have variable degrees of activity against fungi. Some Class I defensins exhibit very low anti-fungal activity. Development of resistance to some defensins is also a potential problem. Hithertofore, there has been only limited study on the effects of defensins on human and animal pathogens.
Defensins with highly divergent sequences act via different mechanism of actions. Permeabilization of the plasma membrane is a common feature that is observed for a number of defensins. However, the mechanism of permeabilization and its role in cell death differs between different defensins. Some defensins cause membrane permeabilization at high concentrations, but not at the concentration required for complete growth inhibition. In fact, the concentration of these proteins required to cause significant membrane permeabilization is around 20 times that required for growth inhibition. These proteins do cause slight membrane permeabilization at concentrations required for growth inhibition but only after long time periods (>150 mins). This probably occurs after fungal cell death. The cell-impermeate nucleic acid SYTOX [trade mark] green assay described in U.S. patent application Ser. No. 12/535,443 has been successfully used as a measure of the rate and extent of permeabilization.
In contrast to some other plant defensins, the plant defensin NaD1 causes significant membrane permeabilization at concentrations corresponding to the IC50. Permeabilization of fungal hyphae by NaD1 begins within 15 minutes and reaches its maximum after 80 minutes (van der Weerden et al. (2010) J Biol Chem 285(48):37513-37520). NaD1 also causes some membrane permeabilization at low concentrations that does not cause growth inhibition (van der Weerden et al. (2008) J Biol Chem 283(20:14445-14452). Difference in the permeabilization kinetics between defensins is likely due to differences in the mechanism of action of the proteins. Hence, there is a role in using permeabilization assays to select appropriate defensins.
There is a need to develop protocols to more effectively manage pathogen infection and infestation in humans, animals and plants. The ability to facilitate this control of pathogens with reduced application of chemical agents or antibiotics or without need for this application altogether would reduce environmental contamination and consequential concerns over carcinogenicity and reduce selective pressures leading to antibiotic resistance.